The site of RNA processing and the spatial relationship between RNA processing and transcription in mammalian nuclei have not previously been established and there has been a long-standing interest in determining these parameters within the cell nucleus. There is evidence for and against the cotranscriptional splicing of precursor mRNAs. A. L. Beyer and Y. N. Osheim (Semin. Cell. Biol. 2, 131 (1991)) have shown that nascent transcripts in Drosophila are associated with spliceosomes, and in some cases, the spliceosome and nascent transcripts can be directly visualized by electron microscopy. In contrast, J. K. Nevins (Annu. Rev. Biochem. 52, 441 (1983)) and others have shown that the unspliced transcripts in mammalian cells can be isolated in the poly(A) fraction, indicating that splicing is posttranscriptional.
Previous studies attempting to determine whether or not mRNA transcripts in mammalian cells are localized at the site of transcription or are free to diffuse within the nucleus after synthesis are contradictory. Those studies which have reported localized loci of RNAs have been unable to establish the significance of these localizations. Highly localized nuclear "tracks" of specific viral mRNAs have been observed by fluorescence microscopy in chromatin-depleted nuclear matrix extracts (J. B. Lawrence, R. H. Singer and L. M. Marselle. Cell 57,493 (1989); Y. Xing and J. B. Lawrence. J. Cell Biol. 112, 1055 (1991)). Several viral RNAs have been observed to localize at only one or two sites within the nucleus (J. B. Lawrence, et al. (1989), supra; J. B. Lawrence, L. M. Marselle, K. S. Byron, J. L. Sullivan and R. H. Singer. Proc. Natl. Acad Sci. USA 87, 5420 (1990); A. Raap, et al. Exp. Cell Res. 197, 319 (1991)), but this observation is in contrast to microinjected globin RNA which appears to diffuse within the nucleus. Lawrence, et al. (1990) speculate that these tracks might represent sites of transcription, but cannot rule out other functions due to the hybridization protocols used. Although these studies demonstrated loci of RNA in the cell, their function could not be determined, e.g., whether they represented sites of transcription, sites of RNA processing, sites of RNA transport, or some other cellular function. Further, they could not spatially correlate the foci to any particular active genes. Total nuclear polyadenylate RNA (poly(A) RNA) has been shown to accumulate in 20 to 50 discrete "transcript domains" which coincide with the location of small nuclear ribonucleoproteins (snRNPs) (K. C. Carter, K. L. Taneja and J. B. Lawrence. J. Cell Biol. 115, 1191 (1991)). These snRNPs have previously been reported to exhibit a clustered nuclear distribution coincident with the spliceosome assembly factor SC-35. Although the association of poly(A) RNA with snRNPs in these studies was observed using hybridization of probes to DNA and mRNA in the same cell, it was not possible to make any conclusions concerning spatial correlation of transcription and processing of a specific RNA with expression of a specific gene because the DNA-specific probe was directed to untranscribed centromere DNA and the rnRNA-specific probe oligo(dT) hybridized to total mRNA.
In the parent application U.S. Ser. No. 07/837,667, cytogenetic preparations were primarily used for the in situ hybridizations. One example (neu oncogene detection) uses fixed cells as in the present invention. The cytogenetic preparation methods degrade nuclear RNA and are therefore not useful in the present invention, which is directed to detection of nuclear RNAs and to simultaneous detection of nuclear DNA and RNA. Although the parent application states that DNA and RNA can be detected simultaneously, this refers only to hybridization under conditions which are permissive for both. The practitioner therefore is unable to distinguish DNA hybridization from RNA hybridization under these conditions. The EBV viral RNA detected in cytogenetic preparations in the parent application accumulates at abnormally high levels in the nucleus and is exceptionally stable. This RNA is produced from a latent viral infection and very little, if any, is transported to the cytoplasm and expressed. This EBV RNA was therefore unusually resistant to the cytogenetic preparation procedure and was detectable using the previously disclosed in situ hybridization methods. It was subsequently discovered, however, that these methods were not suitable for detection of the majority of mRNAs, which are less abundant and less stable than EBV RNA in that particular cell line. The present invention improves and extends those methods to detection of the relatively nonabundant and generally unstable specific nuclear RNAs, which are the products of expression and include most cellular genes and all protein coding genes.
Although the advantages of observing expression of a selected gene in intact cells (i.e., by in situ hybridization) have long been recognized, prior art hybridization methods have been unable to accomplish this, in part due to the lack of available techniques for specific staining of DNA and RNA in the same cell while preserving both the probe signals and the spatial relationship of the transcript and its active gene. The present invention for the first time provides methods for visualizing the intranuclear distribution of specific RNAs correlated to expression of a particular gene, relating this distribution to sites of transcription and processing and identifying larger domains of RNA transcription and processing enriched in polyA RNA and splicing factors. These studies have also established for the first time that mRNA transcripts in mammalian cells are localized at the site of transcription and processing and are not free to diffuse in the nucleus after transcription as some previous studies have suggested. Not only can expression patterns of a selected gene and the distribution of transcripts be identified, the present methods also for the first time allow comparison of the expression of specific alleles of a selected gene (gene imprinting). That is, unless the alleles express RNAs which are sufficiently different to alter hybridization to a probe, conventional in situ hybridization methods which target cytoplasmic RNA (and conventional filter nucleic acid hybridizations as well) are incapable of distinguishing allelic variants at the nucleic acid level. Using probes specific for the maternal or paternal chromosome and the RNA produced by the gene of interest according to the present methods, however, the practitioner can identify expression of an allele spatially correlated to either the maternal or paternal chromosome. Similarly, other genetic mutations which do not affect the rnRNA enough to alter conventional hybridization to a probe may effect the distribution, processing or expression level of the RNA at the cellular level, and these abnormalities may also be detected using the present methods.
Applicants have also demonstrated analysis of the distribution of a specific viral RNA as a means for determining whether an infection is latent or productive. Using the present in situ hybridization methods for detection of EBV-specific RNA, the nucleus of cell latently infected with a single integrated copy of the virus shows a single, very long RNA track present only in the nucleus. A productive infection, in contrast, shows RNA-specific signals in both the nucleus and cytoplasm.
Although techniques for detection of single copy genes by in situ hybridization are available, the low abundance RNAs in the cytoplasm have remained below the limits of detection and sensitivity of non-isotopic methods. It was therefore especially unexpected that the present methods would increase the sensitivity and stability of in situ hybridization to a point where the very low abundance, labile mRNAs which had previously been undetectable in the cytoplasm could be detected in the nucleus. One significant example is dystrophin mRNA (0.01% of mRNA; J. Chelly, J. C. Kaplan, P. Maire, S. Gautron and A. Kahn. Nature 333,858. (1988)), which has been detected by the present methods of in situ hybridization but was previously undetectable in the cytoplasm. The dystrophin gene is carried on the X chromosome, one homologue of which is inactivated, consistent with detection of the dystrophin RNA as a single track. This result may be due to the fact that the instant methods maintain the targeted RNA in highly localized foci or tracks, thus increasing sensitivity by increasing the concentration of nonabundant mRNAs at a given cellular site. The inventive methods may also be used for detection of expression of exogenous gene sequences. The capability of assessing the level of expression of a gene is particularly useful for exogenous genes because they are often not expressed at normal levels and expression levels are often dependent on the site of integration.